1. Field of the Invention
The present invention relates to signal transducing synaptic molecules and particularly to mammalian SYNGAP (Synaptic GTPase Activating Protein), including recombinant SYNGAPs and fragments and derivatives thereof. In one aspect, the invention provides molecules for detecting and analyzing SYNGAPs in vitro and in vivo. In another aspect, the invention provides assays for detecting compounds that modulate SYNGAP or SYNGAP-related activities. The invention has a variety of applications including use in screens to detect pharmacological agents useful in the diagnosis or treatment of disorders associated with SYNGAP.
2. Background
Neurons communicate by a variety of means including synaptic transmission. One form of synaptic transmission involves chemical signaling; a process generally involving neurotransmitter release from one neuron and modulation of a post-synaptic receptor in another neuron. The release and modulation is usually manifested by propagation of a chemical or electrical impulse. See Edelman, G. M. et al. (eds.) (1987) in Synaptic Function, New York: Wiley; and Goodman and Gilman (1996) in The Pharmacological Basis of Therapeutics, 9th ed. J. G. Hardman, et al. (eds) Pergamon Press, NY.
In many instances, chemical signaling requires specialized neuronal structures called chemical synapses. Analysis of chemical synapses has attracted substantial interest. For example, chemical synapses have been reported to be involved in many, if not all, nervous system functions including neuronal plasticity. In particular, neuronal plasticity is believed to impact critical functions such as cognition, e.g., memory and learning, as well as certain neurodegenerative disorders. See e.g., Kandel, E. R. et al; (1991) in Principles of Neuroscience, Appleton and Lange, Norwalk, Conn. and references cited therein.
A variety of approaches have been used in attempts to understand chemical synapses. For example, certain molecular and biochemical approaches have suggested that chemical synapses are structured and include molecules such as receptors, cytoskeletal proteins, and signal transduction molecules. See e.g., Ehlers, M. D., et al. (1996) Curr. Opin. Cell Biol. 8: 484; Sheng, M. (1996) Neuron 17:575; and Huganir, R. L. and Greengard, M. (1990) neuron 5: 555.
More particularly, it has been reported that appropriate chemical synapse structure requires presence of a protein termed PSD-95/SAP90. The PSD-95/SAP90 protein is representative of a family of molecules including SAP120. Specific members of the PSD-95/SAP90 family are localized at or near chemical synapses. See e.g., Ehlers, M. D., et al. supra; Sheng, M supra; Lau, L. F., et al. (1996) J. Biol. Chem. 271:21622; Muller, B. M., et al. (1996) Neuron 17:255; and references cited therein.
There has much effort towards understanding PSD-95/SAP90 and related proteins. For example, it has been reported that most members of the protein family exhibit the same or a closely related structure, i.e., three tandem PDZ (PSD-95, DLG, ZO-1) domains, a SH3 (src homology 3) domain, and an inactive yeast guanylate kinase domain (GK). See e.g., Kim, E., et al. (1995) Nature 378:85; Kornau, H. C., et al. (1995) Science 269:1737.
There has been recognition that PDZ domains exist in proteins associated with cell membranes. For example, it has been reported that the PDZ domains of PSD-95 bind to specific subunits of the (N-methyl-D-aspartate) NMDA receptor. It has also been reported that the last three amino acids of the NMDA receptor subunits define a consensus sequence (T/SXV; X is any amino acid). The subunit sequence has been reported to facilitate the binding. See e.g., Kennedy, M. B. (1995) Trends Biochem. Sci. 20; Gomperts, S. N. (1996) Cell 84:659; Sheng, M. supra.
There has also been recognition that chemical synapses can employ signal transduction to modulate pre- and post-synaptic activity. For example, certain members of the PSD-95/SAP90 protein family are believed to be associated with signal transduction. More particularly, the PSD-95 protein has been proposed to interact with certain signal transduction kinases. That interaction has been proposed to be important to synaptic function. See Gomperts, supra; Saras, J., and Heldin, C. H. (1996) Trends Biochem. Sci. 21:455; Huganir, R. L. and Greengard, M. supra.
There has also been recognition that the PSD-95/SAP90 protein may be capable of serving as an adaptor molecule. More specifically, there have been reports that the PSD-95/SAP90 protein may be able to relate synaptic activity and signal transduction in some instances. That property is believed to important to appropriate synapse function. See e.g., Brenman, J. E. et al. (1996) Cell 84: 757; and Saras, J. and Heldin, Carl-Henrik (1996), supra.
A variety of signal transduction molecules are known including specified kinases and proto-oncogenes. For example, the proto-oncogene Ras is recognized as a G-protein that is apparently involved in signal transduction pathways affecting, e.g., cell growth, cell differentiation, neuronal plasticity and cell survival. In particular, Ras appears to have a substantial role in kinase activation. In addition, biological activity manifested by a variety of neurotrophic factors (i.e. neurotrophins) may be derived through Ras-associated signaling pathways. See e.g., Bokoch, G. M., and Der, C. J. (1993) FASEB. J. 7:750; Marshall, C. J. (1996) Cell Biol. 8:197; Finkbeiner, S. and Greenberg, M. E. (1996) Neuron 16:233; Kang, H., and Schuman, E. M. (1996) Science 273:1402.
Certain neuronal functions have been proposed to be affected by Ras-mediated signal transduction. See e.g., Seger, R., and Krebs, E. G. (1995) FASEB J. 9:726; and Finkbeiner and Greenberg, supra.
Additional signal transduction pathways are known. For example, the inositol triphosphate signaling pathway has been reported to couple modulation of certain receptors to a variety of functions, many of which relate to calcium. See e.g., Berridge, M. J. (1988) Pro. R. Soc. Lond. (Biol) 234: 359.
It would be desirable to identify molecules that impact chemical synapse function and particularly interact with the PSD-95/SAP90 protein. It would be further desirable to identify molecules that can bind the PSD-95/SAP90 protein and affect signal transduction. It would also be desirable to have effective assays for identifying compounds and especially pharmaceutical agents with capacity to modulate the function of these molecules.
The present invention features molecules that relate to SYNGAP (Synaptic GTPase Activating Protein); an excitatory synapse protein that has been found to bind synaptic proteins and modulate signal transduction. In one aspect, the invention provides isolated polynucleotides that encode SYNGAP or fragments or derivatives thereof Further provided are SYNGAP or SYNGAP-related polypeptide encoded by the polynucleotides. In another aspect, the invention provides immunological molecules that are capable of binding the polypeptides. Additionally provided are methods for using the molecules of this invention, e.g., to treat or prevent at least one disorder mediated by SYNGAP. The invention also provides screening assays for detecting compounds useful in the diagnosis or treatment of disorders impacted by SYNGAP.
We have discovered mammalian SYNGAP: a novel protein that binds synaptic proteins important for chemical synapse function. Additionally, we have found that SYNGAP is capable of modulating certain signal transduction molecules and particularly the Ras proto-oncogene (Ras). We have particularly found that SYNGAP is positioned to relate synaptic activity and signal transduction, thereby indicating a significant role in many aspects of nervous system function.
As will be discussed below, SYNGAP is represented by a family of alternatively spliced variants including SYNGAP-a, SYNGAP-b, and SYNGAP-c.
The present invention provides a number of significant uses and advantages. For example, the invention relates, for the first time, recognized synaptic proteins such as PSD95/SAP90 and SAP120 to signal transduction. More particularly, the invention provides SYNGAP and SYNGAP-related molecules that are believed to serve as adaptors between key synaptic proteins and specific transduction pathways. Accordingly, the present invention is expected to facilitate attempts to more fully understand relations between synapses and signal transduction, particularly relationships between synaptic function and signaling molecules such as Ras, inositol triphosphate and certain other transduction molecules.
The present invention has a wide spectrum of important applications. For example, specific molecules of this invention can be used as a diagnostic tool to detect excitatory synapses, i.e., those that include mammalian SYNGAP. Illustrative of such excitatory synapses are those impacting neuronal plasticity, particularly habituation, sensitization, leaning and memory; as well as certain neurological disorders. In addition, the invention can be employed in the diagnosis, treatment or prophylaxis of certain neurological disorders impacted by SYNGAP. As will be discussed in more detail below, exemplary neurological conditions include those specifically affecting awareness and cognition as well as neuronal growth and survival.
Further, the present invention provides a variety of highly useful molecular markers including polynucleotides, polypeptides, and immune system molecules that can be used in many commercial, medical, home or research settings. For example, certain molecules of this invention can be used in screening assays to detect compounds and particularly pharmaceutical agents useful in the diagnosis or treatment of neurological disorders impacted by SYNGAP. Of particular interest are recognized manipulations that can be employed for the identification of small molecules, e.g., synthetic peptides, peptide mimetics, drugs, etc., that can modulate interaction between SYNGAP and the synaptic proteins to which it associates; interaction between SYNGAP and signal transduction molecules or both. Related techniques can be used to screen for small molecules that potentially block or enhance one or all of these interactions. In particular, in vitro screens are provided that can detect SYNGAP antagonists or agonists.
Additional uses include use of SYNGAP polynucleotides to detect SYNGAP expression in desired cells or groups of cells such as tissue or an organ by conventional in situ hybridization methods.
Particularly useful are SYNGAP-binding immune system molecules of this invention such as the antibodies and antigen-binding antibody fragments provided below. For example, the antibodies (monoclonal and polyclonal) can be used alone or in combination with other agents to facilitate identification of excitatory chemical synapses that include SYNGAP. In some instances, it may be desirable to include the antibodies wholly or as part of a therapeutic strategy to monitor or in some cases modulate the excitatory chemical synapses. Preferred molecules of the invention are flexible and can be modified, if desired, to deliver a desired molecule such as a drug, toxin, enzyme or radionuclide optionally through a linker sequence such as a peptide linker sequence. As an illustration, a SYNGAP binding antibody of this invention can be detectably-labeled to identify excitatory chemical synapses in vitro or in vivo.
The SYNGAP and SYNGAP-related molecules of this invention have additional uses and advantages in vitro and in vivo. For example, the molecules can be employed in functional, cellular and molecular assays (e.g., screens) and in structural analysis, including X-ray crystallography, nuclear magnetic resonance imaging (NMRI), computational techniques. Of particular interest are those techniques involving computer-assisted simulation of synapses (i.e. formulation of virtual synapses). Also included are useful techniques for using the SYNGAP and SYNGAP-related molecules as markers that can provide diagnostic imaging of excitatory chemical synapses in vivo. By way of illustration, certain molecules of this invention can be employed to visualize specific neurons and particularly chemical synapses including SYNGAP in the brain of a living patient. In this embodiment, the desired molecule will usually be detectably-labeled with a suitable tag such as a radionuclide or other suitable imaging component known in the field.
Specific SYNGAP and SYNGAP-related molecules of this invention can be provided in a kit form or other convenient form to facilitate manufacture, packaging, dissemination, storage, and/or use of the present invention.
Accordingly, in one aspect, the invention provides isolated polynucleotides (RNA, mRNA, cDNA, genomic DNA, or chimeras thereof) that encode SYNGAP or a fragment or derivative of SYNGAP. Illustrative of such polynucleotides include those encoding a mammalian SYNGAP, e.g., a primate and particularly a rat or a human SYNGAP. In one embodiment, the polynucleotide encodes a mammalian SYNGAP having a molecular weight of between about 100 to about 15 kDa or greater. In another embodiment, the polynucleotide has at least about 70 percent sequence identity to any of the nucleotide sequences shown in SEQ ID NOS. 1, 3, or 5. Such sequence similarity (i.e. about 70%) or greater similarity will sometimes be referred to herein as xe2x80x9csubstantial homologyxe2x80x9d or like term. Specifically preferred polynucleotides of the invention encode the rat SYNGAP shown in any of the SEQ ID Nos. 2, 4, or 6.
In another aspect, the present invention provides an isolated polynucleotide that is capable of hybridizing to the nucleotide sequence shown n SEQ ID Nos. 1xe2x89xa72, 4-5, or 7-8 under moderate stringency hybridization conditions. In a preferred embodiment, the polynucleotide will also hybridize to those specific sequences shown in SEQ ID NOS. 1-2, 4-5, or 7-8 under high stringency conditions. The terms xe2x80x9cmoderatexe2x80x9d and xe2x80x9chighxe2x80x9d hybridization stringency have readily understandable meaning to those of skill in this field. Exemplary stringency conditions are disclosed in the discussion and examples which follow. In one embodiment, the polynucleotide capable of hybridizing to the SEQ ID NO: 1 or under high stringency conditions is between from about 12 to about 50 nucleotides in length. Illustrative of such polynucleotides are oligonucleotide primers made by conventional synthetic methods. In another embodiment, the polynucleotide is between about 100 to about 3500 nucleotides in length or greater. Illustrative of such polynucleotides are restriction enzyme fragments or chemically synthesized fragments complementary to the sequences shown in SEQ ID Nos. 1, 3, or 5.
In another embodiment of the invention, the polynucleotide capable of hybridizing to the nucleotide sequence shown in SEQ ID Nos. 1, 3, or 5 under high stringency conditions has a length of between about 100 up to about 4000 nucleotides or greater. In a preferred embodiment, the polynucleotide is a cDNA encoding an amino acid sequence capable of modulating the proto-oncogene Ras as determined, e.g., by a standard Ras GTPase activity assay. In a specific embodiment, the amino acid sequence encoded by the polynucleotide is capable inhibiting the Ras protein by at least about 10% up to about 100% in the standard Ras GTPase assay. Additionally preferred are polynucleotides that encode polypeptides capable of modulating Ras-mediated signaling transduction pathways and optionally signal transduction molecules xe2x80x9cdownstreamxe2x80x9d of Ras (directly or indirectly). Examples of such downstream molecules include recognized signaling and effector molecules. Illustrative methods for identifying such polynucleotides and amino acid sequences are described below.
One or a combination of standard approaches can be used to monitor Ras GTPase activity in cells or cell lysates. Preferably, the cell or cell lysate will include a naturally-occurring or recombinant Ras protein. Preferred assays for measuring and quantitating the Ras GTPase activity are discussed below.
Additionally preferred polynucleotides of this invention encode a mammalian SYNGAP or fragment or derivative thereof that is capable of significantly reducing inositol triphosphate signaling as determined, e.g., by modulation of phospholipase C activity in a standard phospholipase C enzyme assay. Preferably, the polynucleotide is a cDNA that is capable of increasing or decreasing the enzyme activity by at least about 10% or more up to about 100% relative to a suitable control assay. Specific phospholipase C assays are described below.
Additionally preferred polynucleotides of this invention encode a mammalian SYNGAP or a SYNGAP-related amino acid sequence that is capable of binding at least from about 1, 2 or 3 up to about 10 PDZ domains as determined by a standard PDZ domain binding assay. As noted above, the PDZ domain has been reported to be present in a variety of membrane proteins, e.g., the PSD95/SAP90 and SAP 120 proteins, and is believed to significantly impact chemical synapses.
By the term xe2x80x9cSYNGAP-relatedxe2x80x9d nucleotide or amino acid sequence or similar term is meant a fragment or derivative of SYNGAP sequence (polynucleotide or polypeptide as described below.
A variety of methods are known in this field for detecting and quantifying, if desired, PDZ binding. In general, the methods are capable of detecting formation of a binding complex and can be optimized to provide qualitative or quantitative characterization of those binding complexes. Illustrative methods for detecting the PDZ binding are more fully disclosed in the discussion and examples that follow.
Additionally preferred polynucleotides are those molecules that encode an amino acid sequence that includes at least a Ras GTPase Activating Protein (GAP) domain up to about 2 to about 3 GAP domains, and a C-terminal sequence that includes at least the following general amino acid sequence: (T or S), X V; wherein X is an amino acid, preferably one of the 20 natural amino acids.
Further preferred are those polynucleotides that encode an amino acid sequence that includes at least one pleckstrin homology (PH) up to about 3 PH domains and at least one C2 domain up to about 3 C2 domains.
Particularly preferred polynucleotides of this invention encode an amino acid sequence that includes in an N- to C-terminal orientation: at least one PH homology, preferably one PH homology; at least one C2 domain, preferably one C2 domain; at least one GAP domain, preferably one GAP domain; and the C-terminal sequence having the sequence (T or S), X V described above. Additionally preferred is a polynucleotide encoding an amino acid sequence that includes in an N- to C-terminal direction at least about the following amino acids of SEQ ID NO. 6, 4 to 72, 87 to 190, 266 to 502 and 1132 to 1135.
Specifically preferred are those polynucleotides that encode a rat SYNGAP as represented by any one of SEQ ID NOS. 2, 4 or 6 including fragments or derivatives thereof.
One or a combination of different strategies can be used to analyze the SYNGAP or SYNGAP-related molecules disclosed herein, e.g., to detect homologous molecules or to identify protein domains (e.g., GAP, PH, and C2). Specifically included are biochemical, immunological and biosensor-type assays as well as certain well-known computer-assisted manipulations.
Additionally provided by the present invention are fragments or derivatives of the polynucleotides encoding mammalian SYNGAP or SYNGAP-related molecules, as well as recombinant vectors including the polynucleotides or the fragments or derivatives thereof It is generally preferred that the recombinant vector be capable of propagating the isolated polynucleotide in a suitable prokaryotic or eukaryotic host cell. Additionally preferred recombinant vectors are capable of expressing that isolated polynucteotide as RNA and preferably mRNA, in a suitable cell expression system. The recombinant vector can include nearly any number of useful elements, however in most cases the vector will include control elements operably linked to the inserted nucleic acid (e.g., promoter, leader, and/or enhancer elements) which control elements can be selected to optimize replication and/or transcription of the vector in the cells.
As noted, polynucleotides of the invention generally encode mammalian SYNGAP or a fragment or derivative thereof In one embodiment, the polynucleotides are substantially homologous to the SYNGAP sequence shown in SEQ ID NO: 2, 4, or 6. In a specific embodiment, the isolated polynucleotides include or consists of cDNA and have a length of between about 50 to about 100 nucleotides up to about 4000 nucleotides or more, as determined by standard nucleic acid sizing methods. In another embodiment, the isolated nucleic acid includes or consists of RNA and particularly mRNA that is also substantially homologous to the specific rat SYNGAP sequence and which can have substantially the same length as the cDNA.
Also provided are host cells that include a polynucleotide as disclosed herein including those that express SYNGAP, a SYNGAP-related molecule or a fragment or derivative thereof under suitable cell culture conditions. Preferably, the host cells are capable of expressing the desired amino acid sequence in the host cell, cell medium, or both.
The invention also includes methods for isolating a polynucleotide encoding a mammalian SYNGAP or SYNGAP-related molecule such as the rat SYNGAP sequence specifically described below. In general, the methods include introducing the polynucleotide into host cells, typically as a recombinant vector including the polynucleotide, culturing the host cells under conditions suitable for propagating the polynucleotide and purifying the polynucleotide from the host cells to obtain larger portion of the isolated polynucleotide therefrom. Host cells useful for propagating the polynucleotides and/or polypeptides of this invention can be eukaryotic, prokaryotic, more particularly, fungal, yeast, animal or insect as desired. Host cells amenable for large scale production of mammalian SYNGAP are especially useful for commercial and industrial applications. Alternatively PCR amplification or related amplification methods can be used to isolate the polynucleotide.
Further provided are cultured host cells which have been transformed, transfected or infected either transiently or stably by at least one recombinant vector of the invention which vector includes an isolated polynucleotide that encodes a mammalian SYNGAP or a fragment or derivative thereof (DNA or RNA).
Recombinant vectors of the invention can be introduced into suitable cells or groups of such cells including tissue or organs if desired either in vitro or in vivo. Preferably, the cells are capable of expressing the recombinant vector at detectable levels. Host cells comprising the vectors can be cultured in medium capable of supporting propagation and/or expression of the vectors in the cells. The cells can be eukaryotic cells, preferably mammalian cells such as neurons and neuron-associated cells (e.g., glia) which cells are capable of expressing desired sequences in the recombinant vector. The cells can be primary cells or the cells can be immortalized. In some instances it will be desirable to introduce the vector into a suitable prokaryotic host e.g., bacteria, insect, yeast or fungal cells to propagate the vector.
The present invention also provides useful oligonucleotide primers, typically single-stranded primers, which oligonucleotide primers are complementary to a polynucleotide encoding a mammalian SYNGAP. For example, in one embodiment, the oligonucleotide primers are complementary to the rat SYNGAP sequence shown in any one of SEQ ID Nos. 1, 3, or 5. The oligonucleotide primers have a variety of useful applications, e.g., to detect or amplify a mammalian SYNGAP of interest. Exemplary oligonucleotide primers will generally have length of between about 12 to about 70 nucleotides although somewhat larger or smaller primers are useful for some applications.
Additional polynucleotides of the present invention have important uses. For example, as discussed, the invention provides for recombinant vectors that include an isolated polynucleotide relating to SYNGAP. Specific recombinant vectors can be used to produce significant amounts of nucleic acid sequence that can be sense or anti-sense, single-stranded or double-stranded as needed. Generally, RNA transcribed from DNA is referred to as the xe2x80x9csensexe2x80x9d RNA strand and oppositely oriented RNA is termed antisense RNA. Antisense polynucleotides, then, refer to sequences of DNA or RNA which can bind in a Watson-Crick fashion to a sequence on a target mRNA. See generally Bentley, D. L. and Groudine, M. (1986) Nature 321:702; and Kimelman, D. Gene regulation: Biology of Antisense RNA and DNA, R. P. Erickson, J. G. Izant, eds. (Raven Press, New York).
Mammalian SYNGAP RNA and particulary a SYNGAP mRNA existing in a biological sample such as in an excitatory chemical synapse (in vivo or in vitro) will sometimes be referred to herein as a xe2x80x9ctargetxe2x80x9d to denote potential for specific binding between a polynucleotide of interest, e.g., a suitable anti-sense RNA, and the SYNGAP mRNA in the sample.
In one preferred embodiment, the recombinant vectors include DNA sequences that encode an anti-sense RNA which RNA is substantially homologous to mammalian SYNGAP polynucleotides of this invention e.g., a rat SYNGAP. It is preferred that the recombinant vectors include cDNA sequences. In this instance it will be understood that the anti-sense RNA will usually include a uracil (U) in place of thymidine (T) where the cDNA sequence has a thymidine. In a preferred embodiment, the anti-sense RNA has a length of at least about 20 to about 50 nucleotides, at least about 100 to about 250 nucleotides, at least about 300 to about 700 nucleotides, at least about 1000 to about 2000 nucleotides and up to about 2500 to about 4000 nucleotides as determined by standard polynucleotide sizing methods. In most cases, the length of the anti-sense RNA will be guided by intended use including the length of the target and the level of anti-sense suppression desired.
The antisense RNA encoded by specific recombinant vectors of this invention is usually designed to undergo complementary base pairing (hybridization) with the target, rendering the target essentially unavailable for translation in most cases. In some instances, the antisense RNA will render the target susceptible to degradation, thereby substantially reducing the amount of the target in relevant cells or tissue. Accordingly, the recombinant vectors of the invention can be used to control the synthesis and/or expression of SYNGAP in desired neurons in vitro or in vivo.
Specific recombinant vectors of this invention that are capable of producing anti-sense RNA complementary to a mammalian SYNGAP mRNA (or more than one of such mRNA) can be used therapeutically to reduce levels of the target in vivo or in vitro. For example, in one embodiment, a desired recombinant vector is administered to a patient suffering from or suspected of suffering from a SYNGAP related disorder such as those specified herein. In this instance, the patient will benefit from substantially reduced or totally absent levels of the target. Preferred administration is sufficient to reduce levels of the target mRNA in the patient. Efficacy of the technique can be monitored and quantified if desired by a variety of techniques including Northern and Western blotting.
In another embodiment, the recombinant vector is formatted to produce anti-sense DNA of about the same size as the anti-sense RNA.
In addition, the polynucleotides of this invention and particularly the isolated nucleic acids and recombinant vectors described herein can be used as important controls for detecting and analyzing normal and aberrant mammalian SYNGAP expression in vitro and in vivo.
In another aspect, the present invention provides isolated mammalian SYNGAP preferably having an apparent molecular weight of between about 100 to about 150 kDa. The molecular weight of the SYNGAP can be determined by a variety of standard means including polyacrylamide gel electrophoresis. Preferred are polypeptide sequences having at least about 200 amino acids up to about 1000 amino acids or greater including about 1100 amino acids. Specifically provided is rat SYNGAP as shown at the amino acid sequence level in any one of SEQ ID NOS. 2, 4, or 6.
In another aspect, there is provided an isolated polypeptide having at least about 170 percent amino acid sequence homology to the sequence illustrated in SEQ ID NO: 2, 4, or 6. Additionally provided are isolated fragments or derivatives of that polypeptide.
The invention also provides methods for producing a mammalian SYNGAP in which the method includes culturing the host cell in medium under conditions suitable for expression of the SYNGAP in the host cell or medium.
In another aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of binding the amino acid sequence shown in SEQ ID NO. 6. Preferably, the binding of the antibody or antigen-binding fragment is blocked by at least about 80%, 90% or more up to about 100% by contact with the amino acid sequence shown in SEQ ID NO:21 or a sequence substantially homologous thereto. The percent blocking by the amino acid sequence can be determined by a variety of means including a conventional immunoprecipitation assay or a Western immunoblot.
In one preferred embodiment, the antibody is a capable of binding excitatory synapses as determined by microscopy. In this embodiment, the antibody can be a monoclonal or a polyclonal antibody or an antigen-binding fragment thereof. In most cases, the antibody or antigen-binding fragment will be detectably-labeled or will be capable of generating a detectable label as specified below to help visualize and optionally quantitate synaptic binding.
Additionally, the invention pertains to methods for making specific immune system molecules and particularly antibodies (polyclonal, monoclonal or chimeric molecules) which bind certain SYNGAP or a SYNGAP-related molecules described herein. The methods generally include using a substantially purified sample of a desired SYNGAP polypeptide, as immunogen. Exemplarly antibodies are monoclonal antibodies obtained by conventional hybridoma manipulations. The antibodies can also be generated from an immunogenic peptide that comprises one or more epitopes of the polypeptide. It will be useful in some settings to covalently attach a suitable cytotoxic, anti-metabolic, or detectable label to the antibody by methods well-known in the field to help detect and/or modulate desired synapses that include mammalian SYNGAP and particularly human or rat SYNGAP in vitro or in vivo.
It will be understood that an xe2x80x9cimmune system moleculexe2x80x9d generally relates to antibodies and antigen-binding fragments of those antibodies that are derived from the immune system of a mammal such as a mouse, rat, rabbit, human, and the like.
The immune system molecules of this invention provide a number of uses and advantages. For example, the immune system molecules can be used to modulate mammalian SYNGAP expression in vitro or in vivo, e.g., by employing conventional microinjection techniques. In one approach, a suitable amount of a desired SYNGAP antibody is suspended in a physiologically acceptable buffer and injected into a desired cell or group of cells including tissue or an organ, which amount is sufficient to reduce or eliminate SYNGAP expression as determined, e.g., by the biochemical and functional assays described herein. In some instances, such approaches will be well-suited to modulate SYNGAP function, e.g., to impact neuronal plasticity in vitro or in vivo.
In another aspect, the present invention provides a kit that typically includes at least one container means comprising at least one of: 1) an antibody or antigen-binding fragment thereof capable of binding mammalian SYNGAP, 2) an isolated polynucleotide comprising sequence with at least about 70% sequence homology to any one of the sequences shown in SEQ ID NOs: 1, 3, or 5; 3) a pair of oligonucleotide primers capable of hybridizing to any one of the sequences shown in SEQ ID NOs: 1, 3, or 5, preferably under high stringency conditions; and 4) a polypeptide with at least about 70% to about 100% sequence homology to the sequence shown in any one of the sequences shown in SEQ ID NOs: 2, 4, or 6 a fragment or a derivative thereof.
In one embodiment, the kit includes a system for: 1) treating or preventing a disorder in a mammal associated with the SYNGAP, 2) detecting excitatory synapses in a cell or group of cells in vitro or in vivo, or both.
Isolated polynucleotides and polypeptides of the invention can be obtained as a substantially pure preparation if desired. That is, the nucleic acids and polypeptides can be isolated in substantially pure form by standard methods and can be provided as sterile preparations if desired. Methods for providing substantially pure preparations of nucleic acids and polypeptides are discussed below.
In another aspect of the present invention, there is provided methods for modulating excitatory synapse function in a cell or group of cells in vitro or in vivo. In this embodiment, the method includes administering to the cells a modulation effective amount of at least one polynucleotide of this invention or fragment or derivative thereof By the term xe2x80x9cmodulation effectivexe2x80x9d is meant a change in exciting synapse function (e.g., amout of SYNGAP).
The present invention also includes methods for modulating excitatory synapse function in a cell or group of cells in vitro or in vivo. In this embodiment, the method includes administering to the cells a modulation effective amount of a mammalian SYNGAP of this invention, including a fragment or derivative thereof
In embodiments of the methods for modulating excitatory synapse function, the modulation can include an increase in excitatory synapse number. Excitatory synapses can be visualized and quantified, if desired, by a variety of means including microscopy or centrifugation.
In another aspect, the present invention includes methods for treating a disorder associated with a mammalian SYNGAP including administering to a patient suffering from or susceptible to such disorder an effective amount of at least one isolated polynucleotide of this invention or fragment or derivative thereof
Additionally provided are methods for treating a disorder associated with a mammalian SYNGAP comprising administering to a patient suffering from or susceptible to such disorder an effective amount of an isolated SYNGAP of this invention or fragment or derivative thereof
In embodiments of the methods for treating SYNGAP-related disorders, the disorder can be a neurological disorder of the central (CNS) or peripheral (PNS) nervous system. In a specific embodiment, the CNS disorder is at least one of an affective disorder, a cognitive disorder, or a neurodegenerative disorder. For example, the affective disorder can be depression; the cognitive disorder can be at least one of memory loss, a learning disability, or schizophrenia; and the learning disability can be attention deficit disorder (ADD). In another specific embodiment, the degenerative disorder can be at least one of Parkinson""s disease (PD), Huntington""s disease (HD), senile dementia, or Alzhemier""s disease (AD). In a further specific embodiment, the PNS disorder is amyotrophic lateral sclerosis. In another specific embodiment, the neurological disorder is associated with at least one of trauma, an immune response or ischemia.
The methods for treating or preventing the SYNGAP-related disorders will also find applicability in the manipulation of habituation, sensitization, learning and memory in research settings.
As noted, the present invention provides a variety of screening assays for detecting compounds and especially pharmaceutical agents for use in the diagnosis or treatment of disorders impacted by mammalian SYNGAP.
In one embodiment, there is provided methods for identifying a compound useful in the diagnosis or treatment of a disorder relating to the SYNGAP. In a specific embodiment, the method includes at least one of the following steps:
a) culturing cells capable of forming synapses comprising SYNGAP under conditions conducive to forming or maintaining synapses,
b) contacting the cells with a candidate compound,
c) analyzing the cells for an increase or decrease in the number of synapses; and
d) detecting the increase or decrease as indicative of the compound useful in the diagnosis or treatment of the disorder relating to SYNGAP.
In another embodiment, the invention provides methods for detecting a compound capable of modulating a Ras-activated second messenger pathway. In this embodiment, the method includes at least one of the following steps:
a) providing a Ras response system comprising a recombinant mammalian GTPase Activating Protein at Synapses (SYNGAP),
b) contacting the Ras response system with a candidate compound,
c) analyzing the Ras response system for an increase or decrease in Ras activity; and
d) detecting the increase or decrease in the Ras activity as indicative of the compound capable of modulating the Ras-activated second messenger pathway.
In preferred embodiments, the Ras response system which includes at least one of Ras a nucleotide di- or tri-phosphate; adenylate cyclase; and an isolated polynucleotide encoding the SYNGAP or a fragment or a derivative thereof. The Ras response system can be provided by one or a combination different strategies including being provided in a host cell or a group of host cells (e.g., tissue or an organ), or a lysate of the cells, tissue or organ.
In another embodiment, the invention includes methods for detecting a compound capable of modulating a Ras-Raf (MAP kinase) cascade. The RAS-GTPase activating activity of SYNGAP can be measured using the MAP kinase as a reporter in a suitable primary or cultured cells. In this embodiment, the method includes at least one of the following steps:
a) transfecting a cells with a reporter gene construct capable of being modulated by Ras-Raf (MAP kinase),
b) transfecting the cells with a polynucleotide encoding any one of the SYNGAP sequences shown in SEQ ID Nos. 2, 4, or 6,
c) contacting the cells with a candidate compound; and
d) detecting the reporter gene construct as being indicative of the compound capable of modulating the RasGTPase activity of SYNGAP.
It will be appreciated that in some embodiments of the method, the SYNGAP cDNA can be provided before, during or after transfection of the reporter gene construct.
In another embodiment, the invention includes methods for detecting a compound capable of modulating a phospholipid-activated second messenger pathway. In this embodiment, the method includes at least one of the following steps:
a) providing an inositol triphosphate response system comprising a recombinant SYNGAP,
b) contacting the inositol triphosphate response system with a candidate compound,
c) analyzing the inositol triphosphate response system for an increase or decrease in phospholipase activity; and
d) detecting the increase or decrease in the phospholipase activity as indicative of the compound capable of modulating the phospholipid-activated second messenger pathway. In preferred embodiments, the phospholipase is a phosphoinositide-specific enzyme such as phospholipase C.
In one embodiment of the method, the inositol triphosphate response system further includes at least one of phosphatidylinositol (PI) and an isolated polynucleotide encoding the SYNGAP or a fragment or a derivative thereof In a specific embodiment, the inositol triphosphate response system includes at least one of diacylglycerol, protein kinase C, and inositol 1,4,5 triphosphate (INSP3). In a related embodiment, the phospholipid-activated second messenger pathway is capable of at least one of calcium (Ca+2) release or protein phosphorylation. The inositol triphosphate response system can be provided by one or a combination different strategies including being provided in a host cell or a group of host cells (e.g., tissue or an organ), or a lysate of the cells, tissue or organ.
In another embodiment of the method, at least the pleckstrin (PH) homology of SYNGAP is provided for use with the inositol triphosphate response system. In another embodiment of the method, at least the PH homology and the SYNGAP C2 domain is used with the inositol triphosphate response system.
Additionally provided by the present invention are methods for detecting a compound capable of modulating phospholipid-dependent calcium (Caxe2x88x922) binding to at least the SYNGAP C2 domain. In this embodiment, the method includes at least one of the following steps:
a) mixing a phospholipid, calcium (Ca+2) and at least the SYNGAP C2 domain, the mixing being under conditions conducive to forming a complex,
b) contacting the mixture with a candidate compound,
c) analyzing the mixture for formation of the complex; and
d) detecting the complex as indicative of the compound capable of modulating the phospholipid-dependent binding between the calcium (Ca+2) and at least the C2 domain of SYNGAP.
In another embodiment of the method, at least the PH homology and the SYNGAP C2 domain is used with the inositol triphosphate response system.
In embodiments of the present invention in which use of rat SYNGAP is desired, it will be possible to use a polypeptide sequence that includes and preferably consists of the sequence represented by any one of SEQ ID Nos: 2, 4 or 6. Also contemplated are embodiments of the present invention which include use of a fragment or a derivative of the rat SYNGAP sequence as show in SEQ ID NOs: 2, 4, or 6.
The methods for detecting the compound capable of modulating the phospholipid-activated second messenger pathway are flexible and can be used if one or more components of the inositol triphosphate response system, e.g., PI, act xe2x80x9cupstreamxe2x80x9d or xe2x80x9cdownstreamxe2x80x9d of SYNGAP.
In another embodiment of the present invention, there is provided methods for detecting a test amino acid sequence capable of binding a mammalian SYNGAP. In one embodiment, the method includes contacting the test amino acid sequence with the mammalian SYNGAP or a binding fragment or the derivative thereof under conditions conductive to forming a complex; and detecting the complex as indicative of the test amino acid sequence capable of binding the SYNGAP. For example, in a specific embodiment, the mammalian SYNGAP can be the rat SYNGAP sequence shown in SEQ ID NO: 6 or a suitable fragment or derivative thereof. Detection of the binding can be acomplished by one or a combination of different strategies such as at least one of immunoprecipitation, affinity chromatography, or a suitable biosensor assay.
In another embodiment of the method for detecting a test amino acid sequence capable of binding a mammalian SYNGAP, the contacting step can be performed in cells and the detecting step can include monitoring expression of a detectable gene product expressed by the cells In a preferred embodiment, the steps are performed in yeast cells.
Particulary preferred are those yeast cells (strains) that are suited for performing what is generally known in the field as a xe2x80x9ctwo-hybridxe2x80x9d assay or a related term. In other embodiments, the detecting step of the methods can include screening a polypeptide expression library or a combinatorial peptide library, e.g., by a hybridization type assay using a suitable polynucleotide of this invention, for the test amino acid sequence.
In preferred instances, the methods for detecting the binding between the test amino acid sequence and the SYNGAP will typically register binding between at least one PDZ domain in the test amino acid sequence and a C-terminal sequence in the SYNGAP or fragment or derivative thereof. Preferably, the C-terminal sequence has the following general sequence: (T or S) XV; wherein X is an amino acid as defined below. The SYNGAP sequence used can be the rat SYNGAP sequence disclosed in SEQ ID NO: 2, 4, or 6 as well as suitable fragments or derivatives of that sequence.
Additionally provided are amino acid sequences detected by any of the methods for detecting the test amino acid sequence capable of binding a mammalian SYNGAP.
The present invention includes, in another aspect, methods for detecting excitatory synapses in a cell or group of cells. In one embodiment, the methods include contacting the cells or group of cells with an antibody or antigen-binding fragment of this invention under conditions sufficient to detect the excitatory synapses in the cells or group of cells. In a specific embodiment, the antibody or antigen-binding fragment is detectably-labeled or is capable of producing a detectable label. Illustrative of such labels include a radionuclide; a protein tag; a chromophore; a fluorescent, chemiluminescent or phosphorescent molecule. In another specific embodiment the antibody or antigen-binding fragment thereof is labeled with at least one enzyme capable of producing a chromophore, a fluorescent, chemiluminescent or phosphorescent molecule.
In another aspect, the present invention provides a library that includes a plurality of the polynucleotides or the polypeptides of this invention including fragments or derivatives of those polynucleotides or polypeptides. Illustrative of such libraries include cDNA and genomic DNA libraries, combinatorial and peptide expression libraries.